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Construction of a eukaryotic expression system of HSP65 gene from Mycobacterium tuberculosis, and anti-HSP65 IgG produced in mice

Department of Immunology, Medical School of Wuhan University, Wuhan 430071, PR China

Correspondence Wei Ju juwei512{at}public.wh.hb.cn

Received June 15, 2004
Accepted October 11, 2004

The purpose of this study was to express the HSP65 gene of Mycobacterium tuberculosis in eukaryotic cells and study its primary immune effect in animals. The HSP65 gene was amplified from the H37Rv strain of M. tuberculosis by PCR and then inserted into the expression plasmid pcDNA3.1(–). The recombinant plasmid pcHSP65 was transfected into HeLa cells by using the liposome transfection method and also injected into BALB/C mice to accomplish DNA immunization. The inserted gene was demonstrated to be identical to the reported HSP65 gene sequence. The transfected HeLa cells expressed HSP65 protein; Western blot showed the presence of a 65 kDa band of the inclusion body protein and immunofluorescence testing identified the protein expressed in cytoplasm. Specific IgG for the HSP65 protein could be identified in immunized mice. This study shows that recombinant eukaryotic expression plasmid pcHSP65 was constructed successfully, which lays a foundation for further study of gene therapy.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Tuberculosis is a leading cause of infectious mortality worldwide (Bloom & Murray, 1992). Approximately one-third of the world's population are infected with Mycobacterium tuberculosis (Silva et al., 2001), the causative agent of tuberculosis. Co-infection with HIV and the emergence of drug-resistant strains have led to an increased mortality in tuberculosis cases (Schaaf et al., 1996). Presently the only available vaccine to prevent tuberculosis is BCG (Bacillus Calmette-Guérin), but its protective efficacy ranges from 0 to 85 %. Recent meta-analysis of BCG studies has shown it only provides 50 % protection from pulmonary tuberculosis (Colditz et al., 1994), it has a particularly low efficacy in adults and it cannot be used therapeutically. Since the global situation of tuberculosis has become so severe and BCG is far from being an ideal vaccine (Lamb & Ferns, 2002), it is necessary to search for an efficient and safe vaccine. A new vaccine may be used as a prophylactic and therapeutic vaccine (Johnston & Barry, 1997).

In recent years there have been many reports about DNA vaccines that have induced protection in a number of patients with cancer or pathogen infections; this information indicates that genetic immunization may be a useful alternative method to induce protective immunity against M. tuberculosis (Tascon et al., 1996). Heat-shock proteins (HSPs) have become prominent antigens in triggering humoral and cellular immune responses, which can elicit antibody and effective lymphocyte production (Silva et al., 1995). Mycobacterial HSPs are a type of protein that possesses antigenicity and can enhance the host immune reaction (Quintana et al., 2002). Between 10 and 20 % of all T cells which respond to M. tuberculosis in infected mice are specific for HSP65; this suggests that HSP65 is a major target for T cells during infection. In this study we selected a prominent 65 kDa protein to construct a DNA vaccine that encoded M. tuberculosis antigen HSP65, which is recognized by both humoral and cellular arms of the immune response during M. tuberculosis infection (Chen et al., 2004).

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Production of DNA vaccine.

The HSP65 gene was amplified from genomic DNA of M. tuberculosis H37Rv strain (Institute of Drug and Biological Products Checking, China). The forward primer for PCR was 5'–GC TCT AGA GCA ATG GCC AAG ACA ATT GCG TAC–3' and contained an XbaI site and start code; the reverse primer was 5'–CG GAA TTC TCA GAA ATC CAT GCC ACC CAT G–3' and contained an EcoRI site and stop code. Recovered PCR products were digested by both XbaI and EcoRI, purified, recovered again and orientationally inserted into the eukaryotic expression plasmid backbone pcDNA3.1(–) by standard molecular biology techniques (Sambrook et al., 1989) to yield plasmid pcHSP65. The positive plasmid was transformed into Escherichia coli DH5 competent cells. Then the cloned gene sequence was analysed. The parental vector pcDNA3.1(–) was used as the negative control. DNA for animal immunization was purified, adjusted to a concentration of 1 g l^–1 in physiological saline and then stored at –20 °C until required.

Transfection of HSP65 gene.

HeLa cells (China Center of Type Culture Collection, CCTCC) were cultured in RPMI 1640 medium with 10 % (w/v) FBS (fetal bovine serum) and 1 % (w/v) double antibiotic (penicillin-streptomycin). One microgram purified DNA of pcHSP65 or pcDNA3.1(–) was mixed with LF-2000 reagent (Gibco-BRL) and then the DNA-LF2000 reagent complexes were directly added to HeLa cells and mixed gently. After incubation for 4–6 h at 37 °C in a CO₂ incubator, the growth media were replaced and G418 (0.2 g l^–1) was added, then incubation continued for 48–72 h until ready to assay.

SDS-PAGE and Western blot.

After denaturation in sample buffer containing 2-mercaptoethanol, HeLa cells (equivalent to 1.5x10⁶ cells per lane) were subjected to PAGE with a 5 % (v/v) stacking gel and a 10 % (v/v) gradient gel. Extracts of normal HeLa cells were run in parallel as controls. Following SDS-PAGE separation, proteins were transferred to a nitrocellulose membrane overnight at 15 V. After blocking with 5 % (w/v) dry skimmed milk in TBS at 37 °C for 1 h, membrane strips were incubated with a 1 : 500 dilution of anti-BCG HSP65 mAb (StressGen) at 37 °C for 3 h. Following extensive washing, membrane strips were incubated with a 1 : 200 dilution of HRP-conjugated goat anti-mouse IgG at 37 °C for 1 h, then extensively washed again. Detection was performed by DAB (3,3'-diaminobenzidine). The immune enzymic reaction was stopped by rinsing in distilled water.

Immunofluorescence analysis.

The transfected HeLa cells were washed with PBS three times and collected, centrifuged, then resuspended in PBS. The cells were fixed with 4 % (v/v) paraformaldehyde for 30 min and washed with PBS, then treated with 0.1 % (v/v) Triton X-100 for 10 min. Having been washed with PBS, the HeLa cells were resuspended with 1 : 100 dilution FITC-HSP65 IgG fluorescence antibody while avoiding light for 1 h. The labelled product was diluted by 1 : 5 with PBS and viewed under a fluorescence microscope.

Animal immunization and ELISA.

Twenty-four BALB/C male mice (SPF grade, body weight 16–18 g, 6 weeks old, provided by Medical Laboratory Animal Center of Hubei Province) were divided into three groups randomly. Fifty micrograms of plasmid pcHSP65 was injected intramuscularly in the quadriceps muscle of each hind leg (100 µg total). Control mice were immunized with physiological saline or the parental vector pcDNA3.1(–). Mice were immunized on three occasions at 10 day intervals. Fifteen days after the last immunization, peripheral blood from the tails of the mice was collected and serum was separated.

Specific IgG was detected with indirect ELISA. Each well was coated with 500 ng of Mycobacterium bovis HSP65 StressGen protein overnight at 4 °C. Having been washed with PBS three times, the wells were blocked with 15 % (w/v) calf serum at 37 °C for 1 h. To each well was added 100 µl of 1 : 100 dilution serum sample, and these were then incubated at 37 °C for 30 min. Each sample was tested in triplicate, while positive and negative controls were also used. After washing, all the samples were incubated with 1 : 500 dilution HRP-labelled anti-mouse IgG at 37 °C for 30 min. After further washing 100 µl of substrate solution OPD (o-phenylenediamine) was added to each well, and these were then incubated at 37 °C for 10 min. The reaction was ended with 2 mol l^–1 sulfate acid. The optical density was measured at 490 nm.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Construction of eukaryotic expression plasmid pcHSP65

The M. tuberculosis H37Rv genomic DNA was taken as a template to amplify the target gene through PCR, a specific fragment of about 1642 bp appeared, which was basically identical to that estimated (Fig. 1). The recovered fragments were cloned into eukaryotic expression plasmid pcDNA3.1(–) and transformed into competent E.coli DH5 cells. The target gene was identified to be the expected size by PCR and digestion methods (Fig. 2). The results of DNA sequence analysis were completely identical to the reported gene sequences. This positive recombinant plasmid was named pcHSP65.

View larger version (61K): [in this window] [in a new window]   Fig. 1. Agarose gel electrophoresis of HSP65 PCR product. Lanes: 1, DNA/HindIII markers; 2–4, PCR product; 5, negative control.

View larger version (73K): [in this window] [in a new window]   Fig. 2. Restriction enzyme analysis of pcHSP65. Lanes: 1, DNA/HindIII markers; 2, 3, pcHSP65; 4, pcDNA3.1(–); 5, 6, digested pcHSP65; 7, digested pcDNA3.1(–); 8, PCR product.

HSP65 gene of M. tuberculosis was expressed in HeLa cells

HeLa cells were transfected with the DNA of pcHSP65. The deposition of the cells was collected and analysed by SDS-PAGE and Western blot. The result of SDS-PAGE showed that in the transfected HeLa cellular lytic proteins, a strong specific band was found near to 65 kDa (Fig. 3, arrow). Western blot confirmed that the 65 kDa protein was bound by the mAb of anti-BCG HSP65, demonstrating that the HSP65 gene was expressed in HeLa cells (Fig. 4).

View larger version (111K): [in this window] [in a new window]   Fig. 3. SDS-PAGE analysis of HSP65 expression in HeLa cells. Lanes: 1, non-transfected HeLa cells; 2, HeLa cells transfected with pcDNA3.1(–); 3, HeLa cells transfected with pcHSP65; M, protein markers.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Western blot analysis of HSP65 expression in HeLa cells. Lanes: 1, HSP65 protein of M. tuberculosis; 2, HeLa cells transfected with pcHSP65; 3, HeLa cells transfected with pcDNA3.1(–).

Immunofluorescence assay

Fluorescence appeared in the cytoplasm of HeLa cells that were transfected with pcHSP65 (Fig. 5). The position of fluorescence shows the location of expressed HSP65 protein bound to anti-BCG HSP65 mAb.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Result of gene transfection into HeLa cells. (a) HeLa cells transfected with pcHSP65; (b) HeLa cells transfected with pcDNA3.1(–). Magnification x600.

Animal immunization

An IgG response to HSP65 was observed in mice immunized with pcHSP65. The level of anti-HSP65 antibody was detected with ELISA. Specific IgG was detected 15 days after final immunization; the titre increased to 1 : 1600 at day 60 and then the antibodies stayed at this level stably until day 90. By contrast, no antibody responses were detected in the mice immunized with physiological saline or control DNA of vector pcDNA3.1(–) (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Anti-HSP65 antibody levels in pcHSP65 DNA immunized mice. , pcHSP65; •, pcDNA3.1(–); , saline.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
DNA vaccines have made inspiring progress in some infectious diseases and cancer research (Boyle et al., 1998). The characterization of DNA vaccines which can elicit humoral and specific cellular immune responses has led to this type of vaccine prevailing in research into tuberculosis vaccines (Silva, 1999; Lowrie & Silva, 2000). We selected the prominent and powerful immunogen HSP65 for developing a DNA vaccine to improve host immunity against M. tuberculosis infection.

In this research, the HSP65 gene from M. tuberculosis virulent strain H37Rv was cloned and correctly inserted into eukaryotic expression vector pcDNA3.1(–). In order to identify the target protein expression, the recombinant pcHSP65 was transfected into HeLa cells by liposome mediation and induced to express proteins transiently. Experimental results showed that the HSP65 protein could be expressed in cytoplasm. After that, a primary immunological experiment was performed in animals. High titres of IgG that targeted HSP65 protein were detected in mice injected with pcHSP65. These high titres persisted for a long time, which indicates that this DNA vaccination can induce a specific humoral response in the host. However, whether this DNA vaccine confers levels of protection against M. tuberculosis infection that compare with BCG remains to be elucidated. In conclusion, the present data have demonstrated that a DNA vaccine with HSP65 elicits HSP65-antigen expression in animals and stimulates a specific immune response. Experiments are now in progress to determine the protective efficacy of this vaccine against M. tuberculosis challenge in animals, especially for the cellular response.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This work was partially supported by Wuhan Cheng-Guang Young Science and Technology Programme no. 20025001019 and Hubei Gong-Guan Science and Technology Projects no. 2003AA303B04.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 

Tascon, R. E., Colston, M. J., Ragno, S., Stavropoulos, E., Gregory, D. & Lowrie, D. B. (1996). Vaccination against tuberculosis by DNA injection. Nat Med 2, 888–892.[CrossRef][Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ju, W. Articles by Qu, X. Search for Related Content PubMed PubMed Citation Articles by Ju, W. Articles by Qu, X. Agricola Articles by Ju, W. Articles by Qu, X.

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